Higa M, Shimabukuro M, Shimajiri Y, Takasu N, Shinjyo T, Inaba T
Second Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan.
Diabetes Obes Metab. 2006 Mar;8(2):228-33. doi: 10.1111/j.1463-1326.2005.00488.x.
This study was conducted to clarify cell death and survival signals in pancreatic beta-cell lipotoxicity.
Rat insulinoma INS-1 cells, with or without expression of dominant-negative mutant of Akt (K179M), were cultured with palmitate (C16:0) or oleate (C18:1) and cell numbers were determined by 0.2% eosin dye exclusion assay. The Akt activity was determined by anti-3'-phospho-inositide-dependent protein kinase (Akt)/protein kinase B (PKB) or anti-phospho-Akt (Serine 473) immunoblotting, and nuclear protein nuclear factor-kB (NF-kappaB)-binding activity was by supershift analysis.
Twenty-four hours treatment with palmitate increased the INS-1 cell number at 0.1-0.2 mM but decreased the cell number at 0.5-1 mM. Oleate did not affect cell number at 0.1-1.0 mM. Palmitate dose-dependently increased phosphorylation of 473th serine in Akt/PKB. The K179M form of Akt/PKB abolished palmitate-induced cell proliferation at the low dose and death at the high dose. Nuclear protein NF-kappaB binding was enhanced at 0.2 and 0.5 mM of palmitate but decreased at 1.0 mM.
Results suggest that Akt/PKB signalling is involved in palmitate-induced cell death and survival of pancreatic beta cell.
本研究旨在阐明胰腺β细胞脂毒性中的细胞死亡和存活信号。
将有或无Akt显性负突变体（K179M）表达的大鼠胰岛素瘤INS-1细胞与棕榈酸（C16:0）或油酸（C18:1）一起培养，并用0.2%曙红染料排除法测定细胞数量。通过抗3'-磷酸肌醇依赖性蛋白激酶（Akt）/蛋白激酶B（PKB）或抗磷酸化Akt（丝氨酸473）免疫印迹法测定Akt活性，通过超迁移分析测定核蛋白核因子-κB（NF-κB）结合活性。
用棕榈酸处理24小时，在0.1 - 0.2 mM时增加了INS-1细胞数量，但在0.5 - 1 mM时减少了细胞数量。油酸在0.1 - 1.0 mM时不影响细胞数量。棕榈酸剂量依赖性地增加了Akt/PKB中第473位丝氨酸的磷酸化。Akt/PKB的K179M形式消除了低剂量棕榈酸诱导的细胞增殖和高剂量时的细胞死亡。在0.2和0.5 mM棕榈酸时核蛋白NF-κB结合增强，但在1.0 mM时降低。
结果表明Akt/PKB信号通路参与了棕榈酸诱导的胰腺β细胞死亡和存活。
